Chem. J. Chinese Universities ›› 2010, Vol. 31 ›› Issue (3): 463.

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Activity Analysis of T4 DNA Ligase Based on Linear Fluorescence Probes

LIU Bin, YANG Xiao-Hai, WANG Ke-Min*, TAN Wei-Hong   

  1. State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China
  • Received:2009-11-09 Online:2010-03-10 Published:2010-03-10
  • Contact: WANG Ke-Min. E-mail: kmwang@hnu.cn
  • Supported by:

    国家自然科学基金(批准号: 90606003, 20805012), 国家“八六三”计划项目(批准号: 2007AA022007)和湖南省杰出青年科学基金(批准号: 08JJ1002)资助.

Abstract:

A continuous, simple method with high specificity for analyzing T4 DNA ligase activity is introduced by monitoring the fluorescence change in real time. Linear DNA probes that can occur in fluorescent resonance energy transfer were used as templates and detecting probes in a homogeneous solution. The limit of detection of T4 DNA ligase was 1.2 U/mL, the KM and Kcat were (1.18±0.06) μmol/L and 40 min-1, respectively. In addition, the method was applied to investigate the influence of chemical drugs and metal ions on T4 DNA ligase activity. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also can be applied to the activity assay, kinetics investigation and drug screening of other ligases.

Key words: Linear fluorescence probe; T4 DNA ligase; Real-time monitoring

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